These montages are made from paraffin sections taken from the parietal region, adjacent to those used in

A thin band of very low cellular density (arrow) can be clearly seen at the proximal border of the cortical plate, and this has been interpreted as representing the nascent subplate in a previous study (). However, GAP43, synaptophysin, and vGABA immunostains reveal a wider band displaying intense immunoreactivity for all three, similar to the marginal zone. We interpret this wider band as marking the full extent of the subplate (see text for further discussion). GFAP immunostaining also shows a subtle transition between the intermediate zone, where glial fibers are relatively disordered, and our definition of the subplate, where glial fibers adopt a parallel orientation, which is also seen in the cortical plate. The boundary between the intermediate zone and the SVZ is most clearly seen in the PAX6 immunostained section where immunoreactivity is almost completely confined to VZ and SVZ. Scale bar = 200 μm.<p><b>Copyright information:</b></p><p>Taken from "A Molecular Neuroanatomical Study of the Developing Human Neocortex from 8 to 17 Postconceptional Weeks Revealing the Early Differentiation of the Subplate and Subventricular Zone"</p><p></p><p>Cerebral Cortex (New York, NY) 2007;18(7):1536-1548.</p><p>Published online 26 Oct 2007</p><p>PMCID:PMC2430151.</p><p></p

Functional annotation clustering carried out in DAVID for differentially regulated genes from a cohort of ALS patients and controls, using RNA extracted from blood with LL and TEM GC undergoing PUMA GEP analysis.

<p>In the case of LL, out of 383 clusters, the top 15 are ranked in order of enrichment score (upper section). In the case of TEM GC, out of 439 clusters, the top 15 are ranked in order of enrichment score (lower section). Common clusters between LL and TEM GC are labelled in italic and underlined.</p

Quality control: globin levels in arrays.

<p>Data were GCRMA normalised and levels of 3 representative alpha globin probe sets (A, B and C) and one beta probe set (D) were averaged for each condition. Expression level box plots indicate that in both the cases of alpha and beta globin, TEM and PAX exhibit highest levels of globin mRNA while TEM GC and PAX GC were the lowest, LL levels were generally lower than PAX and TEM, but higher than PAX GC and TEM GC.</p

Selected QC metrics for LL, PAX GC, and TEM GC arrays.

<p>Scale factor (SF), rawQ, background (BG), percentage present (%P), total signal, actin signal, as well as actin 3′ to 5′ ratio are all listed for LL, PAX GC and TEM GC arrays used for this study. Three arrays in total did not pass quality control and were not analysed: one array was extracted by the LL method, one array by TEM GC and one from TEM.</p

Selected QC metrics for PAX and TEM arrays.

<p>Scale factor (SF), rawQ, background (BG), percentage present (%P), total signal, actin signal, as well as actin 3′ to 5′ ratio are all listed for PAX and TEM arrays used in this study. Three arrays in total did not pass quality control and were not analysed: one array was extracted by the LL method, one array by TEM GC and one from TEM.</p

Characteristics of subjects taking part in the study.

<p>Table indicating patient and control IDs used for this study, as well as blood sample extraction date, age, gender, diagnosis and whether patient was treated with Riluzole. Controls that were matched to patients are also indicated (M = male, F = female, R = treated with Riluzole, P = partner, NBLD REL = non-blood relative). Samples from underlined patients and controls were used in PCR validation.</p

Gene expression analysis.

<p>PCA plotted using QLUCORE OMICS Explorer on GCRMA normalised data: each dot represents an array and takes into account the expression levels of every probe set and shows 2 main clusters (A). One tightly clustering ‘globin negative’ cluster includes the following conditions: LL, TEM GC and PAX GC, while a second more variable cluster “globin positive” includes arrays hybridised with PAX and TEM extracted RNA. Focusing on the globin negative group PUMA analysis identified differentially regulated probe sets (ALS patients vs controls) for the following (B; LL, 3047; TEM GC, 3619; PAX GC, 4511). A Venn diagram comparing these lists shows that TEM and PAX share more genes in common, than either with LL, and 142 genes in common between all 3 methods).</p

PCR primer sequences designed for genes validated in this study.

<p>Samples of cDNA were diluted 1∶10, and 2.5 ul were used per PCR reaction for all genes except EHBP1, where 4 µl was used with 5x Brilliant III Ultra Fast SYBR Green QPCR Master Mix (Agilent). In all cases primer concentrations were optimised to 300 mM (S, sense strand; AS, antisense strand)</p

This figure is taken from frozen sections from parietal cortex at 16 PCW

Synaptophysin immunoreactivity () shows clearly different regions in the cerebral wall. The marginal zone is intensely immunoreactive. The cortical plate is less so but immunoreactivity appears densest in layer IV. The subplate is found between the two small arrows and shows moderately dense immunoreactivity. In the intermediate zone, immunoreactivity appears to be expressed by fibers which are radial in the deeper parts but tangential near to the border with the subplate. () Synaptohysin immunoreactivity at a higher magnification at a border between the subplate and the cortical plate. Distinct punctuate staining which may correspond to synaptic terminals is seen in the neuropil sometimes appearing to surround a putative cell body (asterisk). ( and ) NPY and KCC2 immunoreactive neurons in the subplate, the large arrows show where in the subplate they are found. NPY neurons can be bipolar, orientated radially or tangentially, or multipolar, with varicose processes. KCC2 neurons are often pyramidal in shape. Scale bars = 100 μm in , and 20 μm in , , .<p><b>Copyright information:</b></p><p>Taken from "A Molecular Neuroanatomical Study of the Developing Human Neocortex from 8 to 17 Postconceptional Weeks Revealing the Early Differentiation of the Subplate and Subventricular Zone"</p><p></p><p>Cerebral Cortex (New York, NY) 2007;18(7):1536-1548.</p><p>Published online 26 Oct 2007</p><p>PMCID:PMC2430151.</p><p></p

Initial quality control for the 75 microarrays.

<p>Analysis of array data of all 75 samples run was carried out in Affymetrix Expression Console. PolyA (A) and Hyb (B) controls show that the preparation steps were consistent, and that in general all hybridisations were consistent, but that hybridisation of PAX samples differed from the rest. The % present call (C) values were generally consistent in LL, TEM GC and PAX GC samples, but were clearly lower with TEM and PAX gene samples. The scale factor between chips were generally low, except for PAX hybridisations (D) while the relative log expression (E) of all chips were generally similar with a few exceptions, and a slight general increase in PAX samples (FU, fluorescent units).</p